1. Field of the Invention
This invention is related to method of introducing an endophytic fungus into rough bluegrass. Herein, rough bluegrass refers to a plant which is a Pooideae belonging to the genus Poa which is a part of Poeae and includes Poa trivialis and Poa compressa.
2. Description of Related Art
Bluegrasses known in the art include Kentucky bluegrass, Canada bluegrass, annual bluegrass, rough meadowgrass, bulbous meadowgrass, alpine meadowgrass, wavy meadowgrass, wood meadowgrass, Balfour's meadowgrass, swamp meadowgrass, broad-leaved meadowgrass, narrow-leaved meadowgrass, smooth meadowgrass, spreading meadowgrass and flattened meadowgrass.
Of these, rough bluegrass is known by its English name of rough meadowgrass (Japanese name oosuzumenokatabira).
These plants are members of the group Pooideae, and also belong to the genus Poa which is a part of Poeae. For example, rough bluegrass is known as Poa trivialis. Poa trivialis is a plant classified by Clayton et al (Genera Graminium, Grasses of the World, 1986).
Of the many bluegrasses, rough bluegrasses are of particular importance to man, and are widely used in meadow and pasture.
Rough bluegrasses have a wide utility, and as they cover very large areas, they are subjected to damage from disease or pests. Damage due to bluegrass webworm (Japanese name Shibata moth) is severe, and in areas where pesticide sprays have not reached, grass can disappear overnight as soon as the larvae have hatched.
Conventional methods of cultivating and growing grasses include the artificial crossing method, selection method, mutation method, cell fusion method and gene insertion method. Due to recent progress in biotechnology, the cultivation period which previously required 10 years or more, has been reduced to several years. As regards genetic insertion which is a character transformation tequnigue, several techniques exist such as a method using agrobacterium, the electroporation method and the particle gun method, and they are now being applied to a large variety of crops.
However, in the case of grasses which comprise staple crops, it has been pointed out that this genetic insertion is extremely inefficient. In the case of the agrobacterium method it is difficult to infect grasses, so genetic insertion is very difficult. As regards the electroporation method, a regeneration system has to be developed from the protoplast of the grass, and even if such a regeneration is possible, the characteristics of the plant may suffer damage due to culture mutations.
Concerning the particle gun method, since genes are randomly introduced into the plant organism or culture, the plant obtained frequently becomes a chimera.
In the case of grasses including rough bluegrass, cell culture techniques such as cell fusion or genetic insertion require complex operating procedures and as they are not very efficient, there are very few examples where they have successfully been applied at a practical level.
However, there are some wild plants in nature in which filamentous fungi which are internal fungi, i.e. endophytes, live together with the plant. They grow particularly well in the gaps between cells, i.e. the intercellular spaces.
These endophytes, or symbiotic filamentous fungi, not only have no adverse effect on the host plant but in fact provide it with useful substances, and contribute to help withstanding environmental stresses.
Enhancement by endophytes of plant properties is known from the literature, e.g. insect resistance (Siegel et al, 1987, Ann. Rev. Phytopathol. 25: 293-315), disease resistance (Gwinn and Gavin, 1992, Plant Disease 76: 911-914), environmental stress (drought, etc.) resistance (Arachevalta et al, 1989, Agron J. 81: 83-90), and growth enhancement (Latch et al, 1985, N.Z.J. Agric. Res. 28: 165-168). It is particularly well-known that, in perennial rye grass infected with endophytes, these endophytes improve insect resistance due to the repelling substances and alkaloids they produce.
Latch et al in New Zealand are searching for endophytes known as endosafes which have low toxicity to livestock and excellent insect resistance by collecting and studying endophytes in perennial rye grass.
As many of the plants in which these endophytes live have little utility, it is necessary to introduce them into useful grasses. In this regard, attempts have already been made to introduce endophytes into perennial rye grass which is an important pasture grass. The techniques used may be broadly distinguished as artificial crossing and artificial inoculation.
In artificial crossing, useful characteristics are introduced by pollen using a plant infected by an endophyte as mother, but using the conventional method, there were limitations on the species and strains which could be crossed with one another. On the other hand, in artificial inoculation, plants or culture tissues are inoculated with endophytes that have been separated and cultivated.
The artificial inoculation method is capable of introducing a wider range of types, however due to problems of technique regarding cultivation of endophytes, inoculation conditions and conditions of the plant itself, it is limited to perennial rye grass. To increase the infection rate, a method has been reported where callus is used as the plant tissue which is inoculated. However according to this method, it is necessary to develop a plant regeneration system from the callus, hence the method was still limited to perennial rye grass.
The conventional cell cultivation method involves a troublesome procedure and requires considerable training. It was moreover difficult in practical application since culture mutations caused by character transformation or cell fusion had an effect on the characteristic being introduced or on other characteristics.
In the genetic insertion method, it was not possible to introduce specific characteristics if it was not known which genes had an effect on the characteristics and type of a plant.
Characteristics related to complex factors such as environmental stress could not be introduced by techniques such as genetic insertion. Moreover, plants grown by cell culture techniques were often found to exhibit decreased seed fertility. In the case of grasses this led to a decline of yield and was therefore fatal.
In view of the above, growth techniques using endophytes or improvement of characteristics are a totally new approach to solving the above problems.
When endophytes are introduced into plants by artificial inoculation, due to problems in searching for endophytes and cultivation systems, the technique is limited to perennial rye grass and has never been applied to other useful grasses, such as rough bluegrass. In callus inoculation, it is essential to develop a regenerating system of the plant into which the endophyte is to be introduced. Moreover, inoculation conditions had not been developed to increase the rate of infection of the endophyte.
At present, useful endophytes have been found only in perennial rye grass, tall fescue and meadow fescue which constitute a major limitation to their introduction. In particular, these host plants are foreign types, and no endophytes derived from Japanese original plants had yet been found that were adapted to Japanese environmental conditions.